NEW OBSERVATIONS ON MICROBODIES : A Cytochemical Study on CPIB-Treated Rat Liver

The liver of male rats has been studied after CPIB stimulation by using the peroxidase reaction for localizing catalase in hepatic cells. CPIB administration leads to an increase in the number of microbodies, and it is suggested that one mechanism by which microbody proliferation occurs is a process of fragmentation or budding from preexisting microbodies. Reaction product was observed not only within the microbody matrix, but outside the limiting membrane of the microbody and in association with ribosomes of adjacent rough endoplasmic reticulum. This localization of reaction product is interpreted as evidence that catalase after synthesis on rough endoplasmic reticulum may accumulate near microbodies and may be transferred directly into these organelles without traversing the cisternae of the endoplasmic reticulum or Golgi apparatus.     

Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.     

THE DEVELOPMENT OF MICROBODIES AND PEROXISOMAL ENZYMES IN GREENING BEAN LEAVES

The ontogeny of leaf microbodies (peroxisomes) has been followed by (a) fixing primary bean leaves at various stages of greening and examining them ultrastructurally, and (b) homogenizing leaves at the same stages and assaying them for three peroxisomal enzymes. A study employing light-grown seedlings showed that when the leaves are still below ground and achlorophyllous, microbodies are present as small organelles (e.g., 0.3 µm in diameter) associated with endoplasmic reticulum, and that after the leaves have turned green and expanded fully, the microbodies occur as much larger organelles (e.g., 1.5 µm in diameter) associated with chloroplasts. Specific activities of the peroxisomal enzymes increase 3- to 10-fold during this period. A second study showed that when etiolated seedlings are transferred to light, the microbodies do not appear to undergo any immediate morphological change, but that by 72 h they have attained approximately the size and enzymatic activity possessed by microbodies in the mature primary leaves of light-grown plants. It is concluded from the ultrastructural observations that leaf microbodies form as small particles and gradually develop into larger ones through contributions from smooth portions of endoplasmic reticulum. In certain aspects, the development of peroxisomes appears analogous to that of chloroplasts. The possibility is examined that microbodies in green leaves may be relatively long-lived organelles.     

The occurrence and fine structural characterization of microbodies inEuglena gracilis

Summary The fine structure of an organelle morphologically similar to microbodies found in higher plants and animals was studied in cells ofEuglena gracilis fixed simultaneously in glutaraldehyde and osmium tetroxide. These organelles were 0.4 to 0.8 microns in diameter, bounded by a single membrane, and frequently observed in close spatial association with both endoplasmic reticulum and mitochondria. Their finely granular matrices frequently contained membranous cores. Though these organelles were relatively abundant in acetate- and ethanolgrown cells, they were rarely observed in glucose-grown cells, an indication that they play the same role in the metabolism of 2-carbon substrates as do glyoxysomes in higher plants. The presence of these organelles, assumed to be microbodies, is also of considerable interest since catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected.This work was supported in part by grant GY 3804 from the National Science Foundation to L.B.G.     

Ultrastructure of parenchymal leaf cells of different soybean varieties systemically infected with soybean mosaic virus

A comparative study was made of the ultrastructure of parenchyma leaf cells of different soybean varieties systemically infected with soybean mosaic virus (SMV). It has been shown that virus accumulation and formation of virus-specific cylindrical inclusions (CIs) occur in the infected cells, in addition to intracellular changes showing stimulation of lytic processes, such as activation of smooth endoplasmic reticulum and Golgi apparatus, formation of cytoplasmic vacuoles, cytosegresomes, myelin-like bodies, different disturbances in the structure of cell organelles. Many infected cells demonstrated microbodies with invagination in which cylindrical inclusions were often found showing signs of destruction. It is suggested that such microbodies possess autophagic activity towards CIs. A possible relation of the observed virus-induced ultrastructural cell changes with the degree of SMV affection of investigated varieties is discussed     

CYTOCHEMICAL AND DEVELOPMENTAL CHANGES IN MICROBODIES (GLYOXYSOMES) AND RELATED ORGANELLES OF CASTOR BEAN ENDOSPERM

Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix.     

Fine structural and cytochemical analysis of phragmosomes (microbodies) during cytokinesis inAllium root tip cells

Summary The ontogeny and distribution of phragmosomes (microbodies) during cytokinesis inAllium sativum root tip cells have been studied and complemented with a cytochemical analysis of reactivity with diaminobenzidine (DAB). Incubation in different DAB media revealed the presence of catalase but not peroxidase in these organelles, identifying them as a type of microbody associated with the forming cell plate. Only vacuoles, segments of endoplasmic reticulum and portions of the mature walls stained positively with DAB for peroxidase activity. Microbodies begin to appear in the region of the future cell plate as cells enter late anaphase. They exhibit a moderately electron-opaque anucleoid matrix and are continuous with segments of endoplasmic reticulum (ER). Certain observations have led us to consider that certain aspects of plate formation inAllium require the participation of microbodies: (a) their pronounced numerical increase at the onset of plate formation, (b) their intimate association with regions of the plate where vesicle fusion is in progress, and (c) their rapid numerical decline following vesicular fusion and concomitant cell plate formation. The characteristic spatial association observed between microbodies and the plate-forming vesicles may well reflect their mutual involvement in the metabolism of carbohydrates comprising the middle lamella, being coordinated by metabolic activities in the cytosol, mitochondria and dictyosomes.This study was supported in part by NIH training grant HD 174 to Dr.Hewson Swift and the Marquette University Committee on Research Grants 5641 and 5532.     

MORPHOGENESIS AND DEVELOPMENT OF MICROBODIES OF HEPATOCYTES OF RATS DURING PRE- AND POSTNATAL GROWTH

In hepatocytes of fetal rats, cytoplasmic organelles identifiable as microbodies appeared, although only a few of them showed nucleoids and most of them generally had an electronlucent appearance due to the low density of their matrices. Some of these microbodies, especially those lacking the nucleoid, showed a substantial connection with granular endoplasmic reticulum (ER), suggesting that microbodies might be formed from granular ER. Agranular tubular profiles projecting from the surface of microbodies were found with a high frequency in fetal and neonatal rats; however, this phenomenon may not provide crucial evidence suggestive of the derivation of microbodies from agranular ER. Growth and maturation of microbodies are considered to be brought about by an enlargement of these organelles, an increase in their matrices, an appearance and enlargement of the nucleoids, and an increase in the enzyme involved. The specific activity of urate oxidase in the isolated nucleoid fraction was significantly lower in the earlier stages of postnatal growth than later. Increases in the enzyme activity per nucleoid (maturation of the nucleoid), in the number of microbodies containing nucleoids (formation of the nucleoid), and in the size of nucleoids (growth of the nucleoid), may contribute to increases in the enzyme activity of the tissues.     

Ultrastructure of the vegetative cell ofBrassica napus pollen with particular reference to microbodies

Summary The ultrastructure of the vegetative cell ofBrassica napus tricellular pollen grains, just before anthesis with standard chemical fixation, is reported. The vegetative cell may be regarded as a highly differentiated and metabolically active fat-storage cell. It contains many mitochondria with a well developed internal membrane system, starchless plastids, microbodies, lipid bodies, dictyosomes and numerous vesicles thought to originate from the dictysomes. Rough endoplasmic reticulum organized in stacks of cisternae is also spatially associated with certain organelles, mainly lipid bodies, microbodies and plastids. There are also randomly distributed polyribosome areas. The microbodies are mainly polymorphic in shape and are often observed in contact with lipid bodies. The above spatial relationship implies that the microbodies may have a glyoxysomal function. In the late period of vegetative cell maturation, the microbodies are probably involved in the process of glyconeogenesis in which the conversion of lipid reserves to sugar takes place.Abbreviations VC vegetative cell - VN vegetative nucleus - SC sperm cell - M mitochondria - MB microbodies - L lipid body - P plastid - D dictyosomes     

Ultrastructural and Cytochemical Evidence for the Presence of Peroxisomes in the Generative Cell of Magnolia x soulangeana Pollen Grain

The generative cell (GC) development during three sequentialstages of Magnolia x soulangeana pollen grain maturation wasinvestigated by light and electron microscopy. Plastids werenot identified in this cell but mitochondria, Golgi bodies andvesicles as well as rough endoplasmic reticulum profiles werealways present. Microtubules were also present, their numberincreasing and their disposition varying during GC maturation.The most conspicuous components of the GC cytoplasm were themicrobodies. The latter were few in number in the newly formedGC, and the appearance of their matrix was different from laterdevelopmental stages. A clear microbodial proliferation occurredin the GC during an intermediate stage of pollen maturation.Then, the microbody matrix was either fibrillar to granularas in the vegetative cell microbodies or very dense and compact.The polymorphism and size range and the frequent aggregationof these organelles in one or more clusters were also noteworthy.Tilting of semithin sections as well as the analysis of serialsections suggested that a number or enlarged and irregularlyshaped microbodies co-exist with smaller and more sphericalones, the latter probably originating by budding. In the GCof the mature pollen the microbody-like organelles were in generalmore uniform both in shape and size. The cytochemical test ofDAB was positive in the microbodies of both the pollen cells,thus demonstrating their peroxisomic nature. The function ofthe microbodies in the GC is not clear. In this cell, a fewlipid droplets only exist during the first developmental stageand the microbodies were apparently unrelated to any other organelle.Possibly, these are unspecialized microbodies which are paternallytransmitted, but it is not excluded that, temporarily, theymay play some special role during GC maturation.Copyright 1994,1999 Academic Press Peroxisomes, generative cell, pollen maturation, Magnolia x soulangeana Soul.-Bod     

Occurrence of microbodies in chloromonadophycean algae

Microbody-like organelles occur in the cytoplasm of two chloromonadophycean algae,Vacuolaria virescens Cienkowsky andGonyostomum semen Diesing. Microbodies ofVacuolaria andGonyostomum have a granular matrix which lacks a crystalloid core; they are often present in close association with elements of the endoplasmic reticulum. The occurrence of microbodies in other algae is briefly reviewed.     

Structure of filose amoeba <Emphasis Type="Italic">Rhogostoma minus</Emphasis> Belar 1921 (Cryomonadida,Cercozoa) cell

The structure of the unicellular cell of filose amoeba, Rhogostoma minus Belar, 1921, is studied. Zoospores, cysts, and multinuclear plasmodia have not been found. The cell is covered by a thin shell made out of organic matter. Narrow and branched pseudopodia arise from the pseudostome. The vesicle-shaped nucleus, endoplasmic reticulum, microbodies, and Golgi apparatus are of a usual structure. The oval-shaped mitochondria carry tubular cristae. No flagellar apparatus, fibrillar structures, or extrusive organelles have been found. The amoeba feeds on bacteria. The phylogeny of R. minus in regard to other filose amoebas and flagellates is discussed.     

Microbodies und Diaminobenzidin-Reaktion in den Acetat-Flagellaten Polytomella caeca und Chlorogonium elongatum

Summary In two forms of acetate flagellates, the colourless Volvocale Polytomella caeca and the green Volvocale Chlorogonium elongatum, cell organelles can be demonstrated which are ultrastructurally similar to microbodies of higher organisms. The organelles do not have a close association with the endoplasmic reticulum and are located in the peripheral cytoplasm between the elongated mitochondria. In Polytomella they exhibit more or less spherical profiles in section and have a maximum diameter of approximately 0.2–0.25 . In Chlorogonium the organelles occasionally have an elongated shape and are larger than in Polytomella. Employing the electron microscopic cytochemical reagent diaminobenzidine (DAB)/H₂O₂ to localize the microbodial marker enzyme catalase in these organelles, it was found that no accumulation of the electron-opaque product occurs in the microbodies either at alkaline or neutral pH or at room temperature or 37° C. Only the cristae of mitochondria are stained with the DAB reaction caused by cytochrome oxidase and possibly by a cytochrome peroxidase.Organelles of Polytomella caeca containing catalase or cytochrome oxidase can be separated by rate centrifugation of a crude particulate fraction on a sucrose gradient (Gerhardt, 1971). The particles isolated from the peak of catalase activity show the same fine structural characteristics as the microbodies in situ do. But again, there is no detectable staining of these organelles by the DAB/H₂O₂ reaction.The identity of the microbody-like particles in Polytomella caeca and Chlorogonium elongatum with microbodies in general is deduced despite the negative results in cytochemical localization of catalase in these organelles.     

The fine structure of barley aleurone cells

Summary The ultrastructural morphology of both dry and water-imbibed barley aleurone cells is described. The aleurone cell is characterized by the presence of numerous aleurone grains and spherosomes. In addition, it contains organelles typical of other plant cells including structures similar to microbodies, and rough endoplasmic reticulum characterized by the presence of numerous polyribosomes. It is inferred that the morphological specialization of aleurone cells is related to their biochemical specialization.Work supported by National Science Foundation grant GB5863. The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged.     

PEROXIDASE ACTIVITY IN RAT LIVER MICROBODIES AFTER AMINO-TRIAZOLE INHIBITION

The in vivo effects of 3-amino-1,2,4-triazole (AT) on the fine structure of microbodies in hepatic cells of male rats has been studied by the peroxidase-staining technique. Within 1 hr of intraperitoneal injection AT abolishes microbody peroxidase-staining, and the return of staining coincides temporally with the known pattern of return of catalase activity following AT inhibition; this is further evidence that the peroxidase staining of microbodies is due to catalase activity. Peroxidase staining reappears in the microbody matrix without evidence of either massive degradation or rapid proliferation of the organelles. Furthermore, during the period of return of activity, ribosomal staining occurs adjacent to microbodies whose matrix shows little or no peroxidase staining. These observations are interpreted as evidence that (a) catalase is capable of entering preexisting microbodies without traversing the cisternae of the rough endoplasmic reticulum or the Golgi apparatus, and that (b) the ribosomal staining is probably not cytochemical diffusion artifact and may represent a localized site of synthesis or activation of catalase.     

A diploid epithelial cell line from normal adult rat liver with phenotypic properties of 'oval' cells

A diploid epithelial cell line (termed WB-F344) was isolated from the liver of an adult male Fischer-344 rat and the phenotypic characteristics of the cells were studied. These cells measure approximately two-fifths the volume of freshly isolated hepatocytes. They are histochemically negative for glucose-6-phosphatase and weakly positive for gamma-glutamyl transpeptidase. They produce extensive intercellular reticulin fibers which stain immunocytochemically for fibronectin, and they synthesize both alpha-fetoprotein and albumin, but they do not accumulate glycogen particles. Ultrastructurally, they are polygonal cells with numerous intercellular desmosomes and nexus junctions, and they are partially surrounded by basement membrane-like material. Cytoplasmic organelles include few, but sometimes dilated profiles of rough endoplasmic reticulum, lysosomes, abundant free ribosomes, sparse smooth endoplasmic reticulum and Golgi membranes, microbodies, and small, pleomorphic mitochondria. They express A and C isozymes of aldolase, K isozyme of pyruvate kinase, LDH2 to LDH5 isozymes of lactate dehydrogenase, and 'fetal liver'-type alkaline phosphatase isozyme. When compared with the phenotypes of isolated and purified normal hepatocytes, biliary epithelial (ductular) cells and 'oval' cells isolated from livers treated with chemical carcinogens, the phenotypic properties of the liver epithelial cell line in culture most resemble those of the 'oval' cells.     

Interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules--a quadruple fluorescence labeling study.

To study the interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules, we have developed a quadruple fluorescence labeling procedure to visualize all four structures in the same cell. We applied this approach to study cellular organization in control cells and in cells treated with the microtubule drugs vinblastine or taxol. Endoplasmic reticulum was visualized by staining glutaraldehyde-fixed cells with the dye 3,3'-dihexyloxacarbocyanine iodide. After detergent permeabilization, triple immunofluorescence was carried out to specifically visualize mitochondria, vimentin intermediate filaments, and microtubules. Mitochondria in human fibroblasts were found to be highly elongated tubular structures (lengths up to greater than 50 microns), which in many cases were apparently fused to each other. Mitochondria were always observed to be associated with endoplasmic reticulum, although endoplasmic reticulum also existed independently. Intermediate filament distribution could not completely account for endoplasmic reticulum or mitochondrial distributions. Microtubules, however, always codistributed with these organelles. Microtubule depolymerization in vinblastine treated cells resulted in coaggregation of endoplasmic reticulum and mitochondria, and in the collapse of intermediate filaments. The spatial distributions of organelles compared with intermediate filaments were not identical, indicating that attachment of organelles to intermediate filaments was not responsible for organelle aggregation. Mitochondrial associations with endoplasmic reticulum, on the other hand, were retained, indicating this association was stable regardless of endoplasmic reticulum form or microtubules. In taxol-treated cells, endoplasmic reticulum, mitochondria, and intermediate filaments were all associated with taxol-stabilized microtubule bundles.     

Fine-structural characterization of plant microbodies

Summary Morphology and distribution of the relatively less well known organelles of plants have been studied with the electron microscope in tissues fixed in glutaraldehyde and postfixed in osmium tetroxide. An organelle comparable morphologically to the animal microbody and similar to the plant microbody isolated by Mollenhauer et al. (1966) has been encountered in a variety of plant species and tissues, and has been studied particularly in bean and radish roots, oat coleoptiles, and tobacco roots, stems and callus. The organelle has variable shape and is 0.5 to 1.5 in the greatest diameter. It has a single bounding membrane, a granular to fibrillar matrix of variable electron density, and an intimate association with one or two cisternae of rough endoplasmic reticulum (ER). Microbodies are easily the most common and generally distributed of the less well characterized organelles of plant cells. It seems very probable that they contain the enzymes characteristic of animal lysosomes (containing hydrolases) or animal microbodies (containing catalase and certain oxidases). Spherosomes are also possible sites of enzyme activity but are not as common or as widely distributed as microbodies. For this reason it appears likely that the particles designated as plant lysosomes, spherosomes, peroxisomes, etc., in some of the cytochemical and biochemical studies on enzyme localization will prove to be microbodies.Variations in the morphology and ER associations of microbodies in tissues of bean and radish are described and discussed. Crystal-containing bodies (CCBs) are interpreted as a specialized type of microbody characteristic of metabolically less active cells. Stages in the formation of CCBs from microbodies of typical appearance are illustrated for Avena.The general occurrence of microbodies in meristematic and differentiating cells and their close association with the ER suggest that they may play active roles in cellular metabolism. The alterations in their morphology and numbers that are observed in certain differentiating cells suggest further that the enzyme complements and metabolic roles of microbodies might change during cellular differentiation. If so, microbodies could be the functional equivalent of both microbodies and lysosomes of animal cells.NASA Predoctoral Trainee.Public Health Service Postdoctoral Fellow.     

Filaments associated with the endoplasmic reticulum in the streaming cytoplasm of Chara corallina

Perfused Chara cells capable of resuming ATP-dependent cytoplasmic streaming in low free Ca++ solutions have been examined by electron microscopy for myosin-like filaments. Filaments 44 nm in diameter and up to 3 micron in length have been found associated with the endoplasmic reticulum that along with mitochondria, microbodies and dictyosomes from the endoplasm becomes immobilised around the sub-cortical actin bundles when ATP is depleted. Such endoplasmic filaments have not been detected in association with mitochondria or microbodies and they have not been found in the stationary cortex. These filaments are extracted from the perfused cell by ATP unless motility-inhibiting levels of cytochalasin B are present. The filaments are not detectable in cells inactivated in solutions containing high (10(-4) M) Ca++ concentrations even when the Ca++ level is subsequently lowered. Consistent with their being required for motility, cytoplasmic streaming cannot be effeiciently reactivated by ATP in such filament-depleted cells. The possibility is discussed that the filaments contain myosin and that the endoplasmic reticulum with which they are associated has a major role in generating and transmitting the motive force for streaming.     

The mandibular organ of the lobster,Homarus americanus

The lobster mandibular organ is well vascularized and its polygonal cells are arranged loosely around blood vessels and blood sinuses. Numerous mitochondria and microbodies (peroxisomes) give the acidophilic cytoplasm a finely granular appearance, but there is no evidence of secretory granules. The abundant endoplasmic reticulum is almost entirely agranular and occurs in two morphologically distinct forms: tubular and cisternal. The tubular reticulum is randomly distributed and may represent the site of synthesis and transport of the mandibular organ product. The cisternal reticulum is frequently associated with microbodies. Both forms of endoplasmic reticulum proliferate during mid to late premolt. Mandibular organ ultrastructure closely resembles that of cells known to synthesize steroids or lipids, which suggests that this organ may have a similar function. There is no functional evidence of involvement in molt control in Homarus, but ultrastructural and other evidence suggests an analogy with insect corpus allatum.